CN111315370A

CN111315370A - Medicine with anti-inflammatory bowel disease effect and preparation method and application thereof

Info

Publication number: CN111315370A
Application number: CN201880070394.4A
Authority: CN
Inventors: 邓世平; 曹宇; 李志�; 俞云会; 张奎; 张敏洁; 袁高纲; 徐涛; 鱼刚; 江传亮
Original assignee: Suzhou Pharmavan Cancer Research Center Co ltd
Current assignee: Suzhou Pharmavan Cancer Research Center Co ltd
Priority date: 2018-07-28
Filing date: 2018-07-28
Publication date: 2020-06-19
Anticipated expiration: 2038-07-28
Also published as: WO2020024078A1; CN111315370B; EP3756660A1; US20210015780A1; JP2022089845A; US11602514B2; JP7332167B2; EP3756660A4; JP2021501171A

Abstract

The application provides a medicine with an anti-inflammatory bowel disease effect and a preparation method and application thereof, the medicine has a structure shown as a formula I or a formula II, the medicine and pharmaceutically acceptable salts, solvates, prodrugs, tautomers, stereochemical isomers or pharmaceutical compositions thereof have good effect on inflammatory bowel disease, can be used for preparing medicines for treating inflammatory bowel disease, and have important clinical significance and wide application prospect.

Description

Medicine with anti-inflammatory bowel disease effect and preparation method and application thereof

Technical Field

The application belongs to the field of medicines, relates to a medicine with an anti-inflammatory bowel disease effect, and a preparation method and application thereof, and particularly relates to a myricetin derivative, and a preparation method and application thereof.

Background

Inflammatory Bowel Disease (IBD) is a group of chronic nonspecific Inflammatory Bowel diseases with unclear etiology, including Ulcerative Colitis (UC) and Crohn's Disease (CD), the pathogenesis is unknown, but the relevant pathogenic factors found include: inheritance, infection, environmental pollution, diet, intestinal micro-ecology, etc. Inflammatory bowel disease is a common disease in North America and Europe, and the prevalence rate of ulcerative colitis in Europe and America is 240/10 ten thousand, the prevalence rate is 10/10-20/10 ten thousand, the prevalence rate of Crohn disease is 200/10 thousand, and the prevalence rate is 5/10-10/10 ten thousand. The incidence of IBD has also been increasing in japan over the last 30 years. Although China has no epidemiological data of common people, the number of people in the clinic is very obvious in gradual increase in more than ten years. According to statistics of a plurality of hospital cases, the morbidity of the ulcerative colitis is estimated to be 11.6/10 ten thousand, and the morbidity is about 3/10 ten thousand; the prevalence rate of Crohn's disease is 1.4/10 ten thousand, the incidence rate is about 0.4/10 ten thousand, and the actual cases are likely to be more.

Traditional IBD treatment based on aminosalicylic acid, glucocorticoids, immunosuppressive agents, biologicals, etc. is still mainstream at present, and due to complex etiology and poor curative effect, many patients receiving treatment are not relieved, and as high as 80% of patients with crohn's disease and 30% of patients with UC need to be subjected to surgery, a huge medical need in this field is still not met. In recent years, clinical trials and applications of fecal bacteria transplantation have been on the rise, but patient acceptance is low and further exploration is needed. Therefore, finding a safe and reliable medicament for treating Inflammatory Bowel Disease (IBD) which can enter the intestine to play a significant treatment role, effectively reduce ulcer area and relieve patient symptoms, and does not generate drug resistance is still a problem to be solved urgently in clinic.

The Myrica series Myrica (Myricae) Myrica (Myrica) plants in the Myrica family have wide planting area and long history in China. Discussion of traditional Chinese medicine: the waxberry bark is bitter in taste and warm in nature, has the effects of removing blood stasis, stopping bleeding and relieving pain, and is used for treating traumatic injury, fracture, dysentery, gastric and duodenal ulcers, toothache and the like in folk. Myricetin (Myricanol) is a typical macrocyclic biphenyl type cyclic diarylheptanoid compound extracted from cortex myricae, the structure of the myricetin is shown as a formula III, and the myricetin has a unique chemical structure, so that the myricetin is generally concerned in recent years, the research on the pharmacological activity of myricetin is reported at present, the myricetin mainly relates to the aspects of virus resistance, tumor resistance, oxidation resistance, free radical removal, immunoregulation, allergy resistance and the like, and the myricetin has wide potential application.

CN102552243A discloses an application of myricetin and/or myricetin in preparing antitumor drugs, wherein the antitumor drugs include drugs for preventing and/or treating liver cancer, drugs for preventing and/or treating lung cancer, drugs for preventing and/or treating leukemia, drugs for preventing and/or treating stomach cancer or drugs for preventing and/or treating other tumors.

CN105198714A discloses a myricetin derivative, its preparation method and its application in anti-tumor, the myricetin derivative has the following structure:

wherein R is substituted benzyl. The myricetin derivative is obtained by taking myricetin as a raw material and carrying out substitution reaction with substituted benzyl at normal temperature under an alkaline condition, and the myricetin derivative is applied to the preparation of antitumor drugs by introducing para-position F, Cl, Br, CN and NO on hydroxyl at the 5 th position of the myricetin₂Me or OMe substituted benzyl prodrug can be hydrolyzed slowly in vivo to release parent drug to prolong therapeutic effect and action time, and improve bioavailabilityThe bioavailability and the anti-tumor activity are further enhanced, and the myricetin derivative prepared by the method is only limited to the application in the aspect of anti-tumor.

US8940945B2 discloses a method, material and treatment method for reducing Tau protein, providing a myricanol compound, in the examples myricanol derivatives are isolated from plants of the ricinus genus, for use in the treatment of neurodegenerative diseases. The myricetin and derivatives disclosed in this application are also limited to application in neurodegenerative diseases.

At present, no relevant research reports the application of myricetin and derivatives thereof in the aspect related to inflammatory bowel diseases. In order to better apply myricetin to the specific indication, myricetin is modified based on a certain structure-activity relationship, and the modified myricetin derivative aims to increase the activity and/or the pharmacy and can better meet the medication requirements of treating and preventing Inflammatory Bowel Disease (IBD).

Disclosure of Invention

The application aims to provide a medicament with an anti-inflammatory bowel disease effect, and a preparation method and application thereof.

In order to achieve the purpose, the following technical scheme is adopted in the application:

in a first aspect, the present application provides a medicament having anti-inflammatory bowel disease activity, said medicament having a structure according to formula I or formula II:

wherein R is₁And R₂Each independently selected from any one of hydrogen, ethyl, n-propyl, n-butyl, isopropyl, allyl, isobutyl, tert-butyl, benzyl, acetyl, sulfonic group, phosphoric group, benzoic group, benzamido, benzenesulfonic group, pyridine carboxamido, pyrimidine carboxamido or pyrimidine carboxamido;

R₃any one selected from hydrogen, acetyl, sulfonic acid group or phosphoric acid group; and is

Satisfy the following formula I, R₁、R₂And R₃Not simultaneously being hydrogen, R₁、R₂And R₃Not simultaneously being self acetyl, R₁And R₂Is not benzyl at the same time; in formula II, R₁And R₂Not hydrogen or benzyl at the same time.

The drug having anti-inflammatory bowel disease activity provided by the present application can inhibit IBD-associated COX2 protein expression and strongly inhibit PGE₂The capability of the composition can effectively reduce the ulcer area of inflammatory bowel diseases and relieve symptoms, has no obvious toxic or side reaction, and has high safety and reliability.

Compared with the application of myricetin and myricetin derivatives in the aspect of tumor resistance disclosed in the existing method, the medicine with the anti-inflammatory bowel disease effect provided by the application develops a brand-new application, and the application range of myricetin derivatives is greatly expanded.

The COX2 is an inducible enzyme, can be induced by various cytokines and inflammatory mediators, is closely related to inflammation occurrence, and has been reported at present: most COX2 is not expressed under normal physiological state, and the expression level of COX2 is obviously increased under some pathological states (such as gastric ulcer, inflammatory bowel disease, colon cancer and the like), and PGE₂Is an important inflammation index factor downstream of COX 2.

Preferably, the drug has any one of the following structures of compound a-J:

preferably, the medicine structure with the function of resisting inflammatory bowel disease is

Any one of them.

In the present application, myricetin derivatives with 3 structures are preferred, which have higher COX2 protein expression inhibition rate and higher inflammatory factor PGE₂The inhibition rate of the myricetin derivative can reach 100%, and compared with other myricetin derivatives, the myricetin derivative has more excellent anti-inflammatory effect.

In a second aspect, the present application provides a method for preparing a medicament having anti-inflammatory bowel disease activity having the structure shown in formula I, the method comprising:

carrying out substitution reaction on myricetin shown in a formula III and a nucleophilic reagent in the presence of an alkaline reagent to obtain a compound shown in a formula I, wherein the reaction formula is as follows:

Satisfy R₁、R₂And R₃Not simultaneously being hydrogen, R₁、R₂And R₃Not simultaneously being acetyl, R₁And R₂Not benzyl at the same time.

In the present application, the nucleophile is R₁、R₂And R₃Halides or anhydrides having the same groups, etc., which upon reaction can give the structure of formula I, one skilled in the art can select the nucleophile according to the structure of formula I.

Preferably, the basic reagent comprises any one or a combination of at least two of pyridine, triethylamine, N-diisopropylethylamine, N-methylmorpholine, potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide or potassium tert-butoxide, preferably pyridine or triethylamine.

In the present application, the alkaline reagent is not limited to the above-mentioned reagents, and any alkaline reagent that can provide an alkaline environment and is advantageous for the forward progress of the reaction can be used.

Preferably, the molar ratio of myricetin to nucleophile of the compound represented by formula III is 1: 1 (1-6), and may be 1: 1, 1: 2, 1: 3, 1: 4, 1: 5 or 1: 6.

In the present application, the molar ratio of myricetin of the compound of formula III to the nucleophile determines the course of the substitution reaction. Because different nucleophiles have different steric hindrance and electronic effects, R₁、R₂、R₃The substitution reaction occurs in the order of preference: generally, the order of preference is R₁＞R₂＞R₃(ii) a However, when the substituent corresponding to the substituent is an acid halide, the priority is R₃＞R₁＞R₂(ii) a In the reaction process, the mono-substituted or multi-substituted reaction product can be obtained by controlling the feed ratio of myricetin and a nucleophilic reagent.

Exemplary may be: when the reaction sequence is R₁＞R₂＞R₃During the reaction, myricetin and acetic anhydride react in the molar ratio of 1 to 2, and only R is generated₁And R₂Substitution of the position, in which case a substitution is obtained

When myricetin and allyl bromide react, when the molar ratio of the reaction is controlled to be 1: 1, only R occurs at the time₁Substitution of a position to give a compound of

When the reaction sequence is R₃＞R₁＞R₂When myricetin and chlorosulfonic acid are reacted and the molar ratio of myricetin to chlorosulfonic acid is controlled to be about 1: 1, only R is generated at the time₃Substitution of a position to give a compound of

Preferably, the solvent for the substitution reaction comprises any one of dichloromethane, tetrahydrofuran, acetonitrile, pyridine or triethylamine or a combination of at least two thereof.

Preferably, the temperature of the substitution reaction is 0-80 deg.C, such as 0 deg.C, 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C or 80 deg.C etc

Preferably, the time of the substitution reaction is 1 to 30h, and may be, for example, 1h, 5h, 10h, 15h, 20h, 25h, 30h, or the like.

In addition, the application also provides a preparation method of the medicine with the function of resisting inflammatory bowel diseases, which has the structure shown in the formula I, and the method comprises the following steps:

carrying out oxidation reaction on the compound shown in the formula IV in the presence of an oxidant to obtain a compound shown in a formula II, wherein the reaction formula is as follows:

wherein R is₁And R₂Each independently selected from any one of hydrogen, ethyl, n-propyl, n-butyl, isopropyl, allyl, isobutyl, tert-butyl, benzyl, acetyl, sulfonic group, phosphoric group, benzoic group, benzamido, benzenesulfonic group, pyridine carboxamido, pyrimidine carboxamido or pyrimidine carboxamido; and is

Satisfy R₁And R₂Not hydrogen or benzyl at the same time.

Preferably, the molar ratio of the compound of formula IV to the oxidizing agent is 1: 0.5-5, and may be, for example, 1: 0.5, 1: 1, 1: 1.5, 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5, or 1: 5.

Preferably, the oxidant comprises any one of pyridinium chlorochromate, chromium trioxide, ozone, hydrogen peroxide or sulfur trioxide or a combination of at least two of the foregoing.

Preferably, the solvent for the oxidation reaction comprises any one of dichloromethane, tetrahydrofuran, toluene, N-dimethylformamide, N-heptane or dimethylsulfoxide, or a combination of at least two thereof.

Preferably, the temperature of the oxidation reaction is 0-50 ℃, for example, 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ or 50 ℃.

Preferably, the oxidation reaction time is 1-10h, for example, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h or 10 h.

In a third aspect, the present application provides a pharmaceutically acceptable salt of the drug having an anti-inflammatory bowel disease effect as described in the first aspect.

Preferably, the pharmaceutically acceptable salt is a metal salt of a compound of formula I or a metal salt of a compound of formula II.

Preferably, the metal salt comprises any one of a lithium salt, a sodium salt, a potassium salt, a magnesium salt or a calcium salt, preferably a sodium salt or a potassium salt.

In a fourth aspect, the present application provides a solvate of a drug having an anti-inflammatory bowel disease effect as described in the first aspect.

Preferably, the solvate is a hydrate of the compound shown in the formula I, an alcoholate of the compound shown in the formula I, a hydrate of the compound shown in the formula II or an alcoholate of the compound shown in the formula II.

In a fifth aspect, the present application provides a tautomer or stereochemically isomeric form of the medicament as of the first aspect having an anti-inflammatory bowel disease effect.

In the present application, tautomers refer to cis-trans isomerism of a double bond in a chemical structure, and stereochemically isomers refer to R₃Isomerism of each chiral centre in the radical。

In a sixth aspect, the present application provides a prodrug of the drug having an anti-inflammatory bowel disease action as described in the first aspect.

In the present application, a prodrug having an anti-inflammatory bowel disease action is inactive or less active in vitro, and releases an active myricetin derivative after undergoing metabolic changes in vivo, thereby exerting its action.

In a seventh aspect, the present application provides a pharmaceutical composition comprising a drug having an anti-inflammatory bowel disease effect as described in the first aspect.

Preferably, the pharmaceutical composition further comprises an adjuvant.

Preferably, the excipient comprises any one of, or a combination of at least two of, an excipient, a diluent, a carrier, a flavoring agent, a binder, or a filler.

Preferably, the dosage form of the anti-inflammatory pharmaceutical composition includes any one of an oral preparation, an external preparation, or a parenteral preparation.

For example, in the present application, the pharmaceutical composition can be prepared into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, pastes, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like. Typical routes of administration of a compound or pharmaceutically acceptable salt thereof or pharmaceutical composition thereof in the present application include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

In an eighth aspect, the present application provides a use of the drug having an anti-inflammatory bowel disease action according to the first aspect, a pharmaceutically acceptable salt of the drug having an anti-inflammatory bowel disease action according to the third aspect, a solvate of the drug having an anti-inflammatory bowel disease action according to the fourth aspect, a tautomer or stereochemical isomer of the drug having an anti-inflammatory bowel disease action according to the fifth aspect, a prodrug having an anti-inflammatory bowel disease action according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect for preparing a medicament for treating inflammatory bowel disease.

Compared with the prior art, the method has the following beneficial effects:

the drug with the function of resisting inflammatory bowel diseases, and pharmaceutically acceptable salts, solvates, prodrugs, tautomers or stereochemical isomers or pharmaceutical compositions thereof provided by the application can play a good role in inflammatory bowel diseases, and can be developed into a medicine for preventing and treating inflammatory bowel diseases. The medicine with the function of resisting inflammatory bowel diseases has better druggability, can effectively reduce the ulcer area of the inflammatory bowel diseases and relieve symptoms, is safe and reliable, has clinical significance for diseases needing relieving and improving symptoms such as the inflammatory bowel diseases, and has wide application prospect.

Drawings

FIG. 1 shows the NMR spectrum of Compound A of example 1 of the present application.

FIG. 2 is a NMR spectrum of a compound G of example 7 of the present application.

FIG. 3 is a hydrogen nuclear magnetic resonance spectrum of the compound of example 8.

FIG. 4A is a graph showing the effect of Compound A on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

FIG. 4B is a graph showing the effect of Compound B on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

Fig. 4C is a graph of the effect of compound C on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

Fig. 4D is a graph of the effect of compound D on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

FIG. 4E is a graph showing the effect of Compound E on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

FIG. 4F is a graph showing the effect of Compound F on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

FIG. 4G is a graph showing the effect of Compound G on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

Fig. 4H is a graph of the effect of compound H on the proliferative activity of mouse macrophage RAW264.7 in example 10 of the present application.

Fig. 5 is a graph of the effect of compound a-H on the level of IL-6 protein secreted by the mouse macrophage RAW264.7 in example 10 of the present application (n-3).

Figure 6 graph of the effect of compound a-H in example 10 of the present application on the activity of TNF- α protein levels secreted by the mouse macrophage RAW264.7 (n-3).

FIG. 7 PGE secreted by compound A, B, G and H from example 10 of the present application on mouse macrophage RAW264.7₂Influence of protein level (n-3).

FIG. 8 is a graph showing the effect of compounds A, B, G and H on COX2 protein expressed by the mouse macrophage RAW264.7 in example 10 of the present application.

FIG. 9 PGE expressed by Compound H in example 10 of the present application on mouse macrophage RAW264.7₂Activity at protein level is shown in comparison (n-3).

Figure 10 graph of the effect of compound H in example 11 of the present application on the rate of body weight change in the 5% DSS-induced acute IBD model (n-10) (blank group vs 5% DSS p < 0.001; 5% DSS vs 5% DSS +5mg/kg # # p < 0.01).

Figure 11 a graph of the effect of compound H in example 11 of the present application on IL-6 protein levels in serum of a 5% DSS-induced acute IBD model (n-10).

Figure 12 graph of the effect of compound H in example 11 of the present application on DAI score in a 5% DSS-induced acute IBD model (n-10).

Figure 13 graph of the effect of compound H in example 11 of the present application on colorectal pathology in a 5% DSS-induced acute IBD model.

FIG. 14 is a graph of the effect of Compound H in example 11 of the present application on colorectal MPO in a 5% DSS-induced acute IBD model.

FIG. 15 is a schematic representation of the colorectal wet weight changes in rats treated with Compound H of example 12 of the present application.

FIG. 16 is a schematic representation of the area of colorectal ulcers in rats treated with Compound H of example 12 of the present application.

FIG. 17 is a graphical representation of the inflammation score for compound H treatment of rat colorectal pathology HE in example 12 of the present application (p < 0.05vs blank # p < 0.05vs 2.5% TNBS + vehicle).

Figure 18 a schematic representation of compound H treatment of rat colorectal pathology HE gross injury score in example 12 of the present application (. p < 0.05 vs. blank # # p < 0.01 vs. 2.5% TNBS + vehicle).

Figure 19 is a schematic representation of compound H treatment of rat colorectal pathology HE ulcer lesion scores in example 12 of the present application (. p < 0.05 vs. blank # p < 0.05 vs. 2.5% TNBS + vehicle).

FIG. 20 is a schematic representation of compound H treatment of colorectal anatomic lesions in rats according to example 12 of the present application.

FIG. 21 is a schematic representation of the treatment of rat colorectal HE pathology with Compound H of example 12 of the present application.

Detailed Description

The technical solution of the present application is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present application and should not be construed as a specific limitation of the present application.

Example 1

This example prepares 5, 17-diacetoxy myricetin by the following method

Adding myricetin (0.49g, 1.37mmol), dichloromethane (30mL) and pyridine (0.32g, 4.10mmol) into a 100mL eggplant-shaped bottle in sequence under the protection of nitrogen; and diluting acetic anhydride (0.31g, 2.20mmol) with dichloromethane (20mL), placing in a constant pressure dropping funnel, slowly dropping into the eggplant-shaped bottle, and reacting for about 30 min. The system is transferred into an oil bath at 60 ℃ for reflux reaction for 3 hours, and the reaction liquid is taken for LC-MS detection. The reaction solution was subjected to gradient elution by preparative liquid chromatography (mobile phase: petroleum ether-ethyl acetate: 0-35%), the received solutions were combined, and the solvent was dried by spinning to give 5, 17-diacetoxy myricetin (white solid A, 0.35g, LC-MS: M/z. RTM.443.1 (M +1)⁺Purity 99.36%), yield 58%. The hydrogen nuclear magnetic resonance spectrum is shown in FIG. 1.

Example 2

This example prepares 5-n-propoxyberry alcohol by the following method

Under the protection of nitrogen, myricetin (0.36g, 1.01mmol) and N, N-dimethyl are sequentially addedDimethylformamide (10mL), 1-chloropropane (0.17g, 2.21mmol) and potassium carbonate (0.42g, 3.01mmol) were placed in a 100mL round bottom flask. The system is reacted in oil bath at 100 ℃ for 3h, and the reaction liquid is taken for LC-MS detection. The reaction solution was subjected to gradient elution using preparative liquid chromatography (mobile phase: petroleum ether-ethyl acetate: 0-35%), the received solutions were combined and the solvent was dried by spinning to give 5-n-propoxyberry alcohol (white solid B, 0.28g, LC-MS: M/z 395.6(M-H)^-99.38%), yield 70%.

Example 3

This example prepares 5-benzyloxy myricetin by the following method

Myricetin (0.36g, 1.00mmol), acetonitrile (10mL), benzyl bromide (0.38g, 2.21mmol) and potassium carbonate (0.42g, 3.01mmol) were added to a 100mL eggplant-shaped bottle in this order under nitrogen protection. The system is reacted for 6 hours in an oil bath at 80 ℃, and the reaction liquid is taken for LC-MS detection. Separating the reaction solution by preparative liquid chromatography (mobile phase: petroleum ether-ethyl acetate: 0-35%) gradient elution, mixing the receiving solutions, and spin-drying the solvent to obtain 5-benzyloxy myricetin (white solid C, 0.20g, LC-MS: M/z ═ 447.6(M-H)^-98.09%), yield 62%.

Example 4

This example prepares 5, 17-Diallyloxypyruvinol by the following method

Myricetin (0.36g, 1.00mmol), acetonitrile (10mL), allyl bromide (0.27g, 2.21mmol) and potassium carbonate (0.42g, 3.01mmol) were added to a 100mL eggplant-shaped bottle in this order under nitrogen protection. The system is reacted for 6 hours in an oil bath at 80 ℃, and the reaction liquid is taken for LC-MS detection. Separating the reaction solution by preparative liquid chromatography (mobile phase: petroleum ether-ethyl acetate: 0-35%) gradient elution, mixing the receiving solutions, and spin-drying the solvent to obtain 5, 17-diallyloxymyricetin (white solid D, 0.35g, LC-MS: M/z 439.6(M-H)^-99.70%), yield 79%.

Example 5

This example prepares 5-allyloxy myricetin by the following method

Myricetin (0.36g, 1.00mmol), acetonitrile (10mL), allyl bromide (0.13g, 1.10mmol) and potassium carbonate (0.42g, 3.01mmol) were added to a 100mL eggplant-shaped bottle in this order under nitrogen protection. The system is an oil bath at 60 DEG CAnd (5) performing a reaction for 6 hours, and taking a reaction solution to be detected by LC-MS. Separating the reaction solution by preparative liquid chromatography (mobile phase: petroleum ether-ethyl acetate: 0-35%) gradient elution, mixing the receiving solutions, and spin-drying the solvent to obtain 5-allyloxy myricetin (white solid E, 0.22g, LC-MS: M/z: 399.2(M + H)⁺96.64%), yield 55%.

Example 6

This example prepares 5, 17-allyloxy myricetin by the following method

5, 17-Diallyloxypyruvinol (1.20g, 2.74mmol) is dissolved in dichloromethane (50mL), pyridinium chlorochromate (2.95g, 13.69mmol) is added, and the mixture is reacted for 1h under the protection of argon. TLC (PE: EA 3: 1) showed disappearance of starting material, reaction was stopped and concentrated to a light brown solid. Subjecting to flash chromatography (petroleum ether-ethyl acetate: 0-35%) gradient elution to obtain 5, 17-allyloxy myricetin (white solid F, 0.71g, LC-MS: M/z 436.6(M + H)⁺94.53%), yield 59%.

Example 7

This example prepares 5-acetoxymyricetin by the following method

Under the protection of nitrogen, myricetin (0.49g, 1.37mmol), dichloromethane (30mL) and pyridine (0.32g, 4.10mmol) are added into a 100mL eggplant-shaped bottle in sequence; and diluting acetic anhydride (0.16g, 1.10mmol) with dichloromethane (20mL), placing in a constant pressure dropping funnel, slowly dropping into the eggplant-shaped bottle, and reacting for about 30 min. The system is transferred into an oil bath at 60 ℃ for reflux reaction for 3 hours, and the reaction liquid is taken for LC-MS detection. Subjecting the reaction solution to gradient elution with preparative liquid chromatography (mobile phase: petroleum ether-ethyl acetate: 0-35%), mixing the received solutions, and spin-drying the solvent to obtain 5-acetoxymyricetin (white solid G, 0.32G, LC-MS: M/z 400.7(M + H)⁺94.67%), yield 80%. The hydrogen nuclear magnetic resonance spectrum is shown in FIG. 2.

Example 8

This example prepares 11-sulfonyloxy myricetin by the following method

Myristyl alcohol (0.36g, 1.00mmol) was dissolved in dichloromethane (20mL) and triethylamine (0.24g, 3.01mmol) was added. In an ice bath, chlorosulfonic acid (0.18g, 1.51mmol) and dichloro-benzene were added dropwiseAnd (3) dripping the solution of methane (10mL) for 15min, and reacting for more than 16 h. TLC (PE: EA 3: 1) showed disappearance of starting material. Adding silica gel (100 meshes, 200 g), mixing, performing column chromatography (mobile phase: dichloromethane-MeOH: 0-15%) by flash preparative chromatography, and spin-drying to obtain 11-sulfonyloxy myricetin (white solid H, 0.38g, LC-MS: M/z: 436.7(M-H)^-97.82%) and yield 86%. The hydrogen nuclear magnetic resonance spectrum is shown in FIG. 3.

Example 9

This example prepares 5, 17-diacetoxy myricetin by the following method

5, 17-diacetoxy myricetin (0.36g, 1.00mmol) is dissolved in dichloromethane (10mL), pyridinium chlorochromate (1.29g, 6.00mmol) is added, and the reaction is carried out for 1h under the protection of argon. TLC (PE: EA 5: 1) showed disappearance of starting material, reaction was stopped and concentrated to a light brown solid. Subjecting to flash chromatography (petroleum ether-ethyl acetate: 0-35%) gradient elution to obtain 5, 17-diacetoxy myricetin (white solid J, 0.32g, LC-MS: M/z 463.2(M +23)⁺94.53%), yield 94%.

Example 10

In this example, compounds A-H prepared in examples 1-8 were tested for anti-inflammatory activity in vitro

The myricetin derivative stimulates and induces bacterial Lipopolysaccharide (LPS) to secrete IL-6, TNF- α and PEG (polyethylene glycol) secreted by mouse macrophage strain RAW264.7₂The expression of isoinflammatory factors influences: inoculating the prepared cell suspension into 96-well plate at 15000 cells/well and 100 μ l/well, standing at 37 deg.C and 5% CO₂Culturing overnight in an incubator, collecting cells by 80%, discarding original culture solution, adding 100 μ L of prepared test solutions with different concentrations into each hole, adding LPS solution after 1h of drug culture, and the final concentration of LPS in the culture system is 100 ng/mL. Parallel 3 wells were set for each concentration, and a cell control (no drug, no vehicle) and a blank medium control (no cells, zero-adjusted wells) were set for further 24 h. The cell culture supernatant is aspirated by using R&D ELISA detection kit (M6000B; MTA 00B; VAL601) for detecting IL-6, TNF- α and PEG of each group₂Expressing inflammatory factors, and collecting cell eggsIn white, each group was examined for COX-2 protein expression using the Waters Blot method.

Figure 4 shows the effect of each myristyl alcohol derivative (i.e. compounds a-H) on the proliferative activity of mouse macrophage RAW 264.7. As can be seen from the results in fig. 4, some of compounds a (40 μ M), B (30 μ M), C (30 μ M), D (30 μ M), E (30 μ M), F (80 μ M), G (40 μ M), and H (80 μ M) had no significant effect on the proliferative activity of mouse macrophage RAW264.7 (n ═ 3), and the subsequent anti-inflammatory activity was performed at this drug concentration or below.

In the cell experiment, n is the experiment times, and each time, 3 multiple wells (repeat number); in animal experiments, n is the number of animals per group (sample size).

Fig. 5, 6 and 7 show the anti-inflammatory activity effect of myricetin derivatives on mouse macrophage RAW264.7 (n-3).

As can be seen from the results in FIG. 5, all myricetin derivatives exhibit the IL-6 inhibitory effect on LPS-induced secretion of RAW264.7 cells, and some compounds such as A, B, G, H have a strong inhibitory effect on LPS-stimulated IL-6 secretion of RAW264.7 cells, indicating that they may have an inhibitory effect on inflammation in serum or intestinal tract of patients with inflammatory bowel disease.

As can be seen from the results in FIG. 6, a part of myricetin derivatives show significant inhibition effect on TNF- α secreted by mouse macrophage RAW264.7 induced by LPS, which indicates that the myricetin derivatives may have inhibition effect on inflammation of serum or intestinal part of patients with inflammatory bowel diseases.

As can be seen from the results of FIG. 7, a portion of myricetin derivative (A, B, G, H) has significant PGE secretion from LPS-induced mouse macrophage RAW264.7 cells₂The inhibition effect indicates that the compound has the inhibition effect on inflammation of human serum or local intestinal tract of inflammatory bowel disease.

As can be seen from the results of fig. 8, myricetin and its partial derivative (A, B, G, H) can inhibit protein expression of COX2 associated with IBD, and the inhibitory effect of derivatives G and H is better than that of myricetin.

As can be seen from the results in FIG. 9, myricetin and its derivative AB, G and H have strong function of inhibiting PGE (inflammatory factor)₂The ability of the derivative H to inhibit PGE can be seen from the figure₂The activity of the compound has good dose dependence, and the inhibition effect of the derivative H is better than that of myricetin under the dosage of 20 mu M.

PGE secreted by mouse macrophage RAW264.7 induced by LPS by myricetin and compound H₂The inhibitory effect is shown in table 1 below:

TABLE 1

	PGE ₂(pg/mL)
Control group	-407.37±57.67
LPS	1154.90±84.20
LPS+H-10μM	804.30±18.46
LPS+H-20μM	604.66±97.52
LPS+H-30μM	448.16±55.26
LPS+H-40μM	289.30±109.41
LPS+H-60μM	-82.01±357.30
LPS+H-80μM	-338.14±138.40
LPS + myristyl alcohol-20. mu.M	904.98±18.02

Example 11

In this example, myricetin derivative compound H was tested for pharmacodynamic activity in vivo in a Dextran Sodium Sulfate (DSS) -induced IBD mouse in vivo model

The method comprises the steps of adaptively feeding SPF grade C57BL/6 mice, female mice and 6-8 weeks old mice for 3-7 days, feeding the mice with 5% Dextran Sodium Sulfate (DSS) aqueous solution, feeding the mice with free drinking water (day0), changing fresh DSS aqueous solution every 2 days, and feeding the mice with fresh purified water after free drinking DSS aqueous solution for 7 days (day 7). After 4 days of molding, the drug was administered to animals at 5 days of molding, and 5mg/kg, 2.5mg/kg and 1.25mg/kg of the drug were administered to the animals as tail vein drug administration. The administration was continued for 4 days, followed by 1 day observation, and the experiment was terminated 24 hours after the last administration.

Detection indexes are as follows: and observing and recording the body weight of each group of animals every day, simultaneously collecting the excrement of each animal, detecting the occult blood condition of excrement, if the obvious excrement can not be judged, confirming the occult blood condition by using an occult blood detection kit, recording the occult blood condition on an experimental recording paper, and taking a picture for recording. At the end of the experiment, animal sera were collected and assayed for expression of IL-6 in the blood. And simultaneously scoring the fecal characteristics, occult blood condition and weight change rate of each animal, and adding the obtained scores to obtain the DAI score of the animal. Animal colorectal tissue samples were collected for subsequent pathology and detection of MPO, etc. within the tissues.

As can be seen from the results of fig. 10, myricetin derivative H showed a reduced weight loss in the 5% DSS-induced mouse acute IBD model. It is indicated that it has the effect of improving the weight loss of patients with inflammatory bowel disease.

As can be seen from the results of fig. 11, myricetin derivative H shows a significant anti-inflammatory effect, indicating that it has an effect of improving inflammatory responses in patients with inflammatory bowel diseases.

As can be seen from the results of fig. 12, myricetin derivative H showed a significant effect of reducing the DAI score of mice, indicating that it has an effect of improving the overall disease state of inflammatory bowel disease patients, and improving the symptoms.

As can be seen from the results in fig. 13, myricetin derivative H showed a significant effect of repairing 5% DSS on intestinal mucosa injury of C57BL/6 mice, indicating that it has an effect of improving intestinal injury of patients with inflammatory bowel disease.

As can be seen from the results in fig. 14, myricetin derivative H has certain effect of improving the oxidative damage of mouse colorectal region caused by 5% DSS.

Example 12

In this example, compound H was tested for pharmacodynamic activity in vivo in a rat model of 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) induced IBD.

The test method comprises the following steps: the method comprises the following steps of adaptively feeding 180-200 g of SPF SD rats to female animals for 5-7 days, starting model building, firstly anaesthetizing the rats during model building, enabling a cat catheter marked in advance to penetrate through anus to enter 8 cm of colon, slowly injecting 250 mu L of 2.5% (wt/vol) TNBS solution into the inner cavity of the colon, inverting the rats for 1 minute, slowly taking out the catheter from intestinal tract after 1 minute, placing the rats with heads facing downwards for 1 minute, then placing the rats back into a cage, and administering the same volume of physiological saline to the rats in a blank group. The next day of molding, D1, was administered by gavage for 6 consecutive days.

Detection indexes are as follows: and observing and recording the body weight of each group of animals every day, collecting excrement of each animal at the beginning of an experiment, detecting the occult blood condition of excrement, if obvious excrement blood cannot be judged, confirming the occult blood condition by using an occult blood detection kit, recording the occult blood condition on an experiment recording paper, and taking a picture for recording. And when the experiment is finished, scoring the fecal characteristics, occult blood condition and weight change rate of each animal, and adding the obtained scores to obtain the final DAI score of each animal. Animal colorectal tissue samples were collected and weighed wet for subsequent pathology testing.

The experimental results show that: the intestinal wet weight of rats after 2.5% TNBS treatment is increased compared with that of the blank group and the treatment group, but no statistical difference exists, but the H-12.5mg/kg administration group has the tendency of reducing the edema of the rats (shown in figure 15).

The experimental results are as follows: oral administration of 12.5mg/kg-H significantly reduced the area of 2.5% TNBS-induced IBD model colorectal ulcers (FIG. 16), which was slightly better than 5-aminosalicylic acid (5-ASA).

The experimental results are as follows: oral administration of 12.5mg/kg-H had a tendency to significantly reduce the level of colorectal inflammation in the IBD model induced by 2.5% TNBS, at which point the efficacy was comparable to that of 5-aminosalicylic acid (5-ASA) (FIG. 17).

The experimental results are as follows: oral administration of 12.5mg/kg-H significantly reduced the level of 2.5% TNBS-induced colorectal gross injury in the IBD model, an index comparable to 5-aminosalicylic acid (5-ASA) (FIG. 18).

The experimental results are as follows: oral administration of 12.5mg/kg-H significantly reduced the 2.5% TNBS-induced lesion score in colorectal ulcers in IBD model, and was slightly more efficacious in this index than 5-aminosalicylic acid (5-ASA) (FIG. 19).

The experimental results are as follows: oral administration of 12.5mg/kg-H, 25mg/kg-H had the effect of reducing 2.5% TNBS-induced lesions in colorectal ulcers in a model IBD (FIGS. 20 and 21).

Pathological observation results show that: oral administration of 12.5mg/kg-H has the effects of remarkably relieving the colorectal inflammation score of 2.5% TNBS-induced IBD model rats (shown in figure 17), remarkably reducing the overall colorectal injury degree of 2.5% TNBS-induced IBD model rats (shown in figure 18), and remarkably relieving the injury severity degree of 2.5% TNBS-induced IBD model rats (shown in figure 19, figure 20 and figure 21).

The above experimental results show that myricetin derivative compound H has good anti-inflammatory bowel disease effect.

Example 13

In this example, compound A, B, G and compound H were tested for acute toxicity by the following specific steps:

the experimental method comprises the following steps: 30 SD rats with half of each male and female are adopted, the weight average of each group is 160-200 g female and 180-220 g male, and the weight of each individual is within +/-20% of the average weight. The animals are allowed to acclimatize for at least 5 days before the test, and healthy (female need not be pregnant) rats are selected as the test animals. Main examination contents of the adaptation period: whether the quality index is consistent with the quality index required in ordering; checking a general state; whether the body weight reaches the body weight range required by the test. Abnormal animals that failed were not included in the trial. Rats are orally taken for a single time, low, medium and high doses are given, the doses are respectively adjusted to be 100mg/kg, 300mg/kg and 1000mg/kg according to a blank preparation pre-experiment, a control group is additionally arranged, and an equal volume of solvent is orally taken.

The observation method comprises the following steps: (1) general state observation: observing the appearance signs, administration parts (bleeding, swelling, bruise, induration, suppuration and fester), hair, general behavior and activity, mental state, gland secretion, skin and mucosa color, respiratory state, fecal character, genital, death and other toxic symptoms of the rat; observing for 1 time about 0-2 hours and 4-6 hours after each administration; the number of observations can be increased if there are toxic symptoms. (2) Gross anatomical observation: all the surviving rats in each group were dissected and observed on the 14 th day of the experiment, and abnormal organ tissues which may be related to the test article were observed and recorded by photographing for the administration site and the general anatomy. (3) Disposal of moribund animals: the status of the rats and the observation time were recorded and the body weight was measured. (4) Treatment of dead animals: the death time or death time of the rats was recorded, and the rats were dissected rapidly after measuring body weight for gross observation and the cause of death was presumed. The acute toxicity doses for compound A, B, G and H and the results are shown in table 2 below:

TABLE 2

The experimental results are as follows: the SD rat has no obvious toxic or side effect after single intragastric administration of the compound, and has no obvious weight reduction and diet reduction trend. The single acute toxicity tolerance MTD of the myricetin derivative is more than 1000mg/kg, wherein the MTD of H is more than 1500 mg/kg.

By combining the above embodiments of the present application, it can be seen that the drug with anti-inflammatory bowel disease effect of the present application, the synthetic raw materials are taken from natural products, which is green and environment-friendly, and the synthetic process is simple and easy to control; from the pharmacodynamic experiment, the compound can be widely used as a medicament for treating inflammatory enteritis (IBD) diseases, has no obvious side effect, and has wide application prospect.

The applicant states that the present application illustrates the drug having anti-inflammatory bowel disease effect of the present application and the preparation method and application thereof by the above examples, but the present application is not limited to the above process steps, i.e., it does not mean that the present application has to rely on the above process steps to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitution of selected materials for the present application, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present application.

Claims

A drug having an anti-inflammatory bowel disease effect, which has a structure represented by formula I or formula II:

wherein R is₁And R₂Each independently selected from any one of hydrogen, ethyl, n-propyl, n-butyl, isopropyl, allyl, isobutyl, tert-butyl, benzyl, acetyl, sulfonic group, phosphoric group, benzoic group, benzamido, benzenesulfonic group, pyridine carboxamido, pyrimidine carboxamido or pyrimidine carboxamido;

R₃any one selected from hydrogen, acetyl, sulfonic acid group or phosphoric acid group; and is

Satisfy the following formula I, R₁、R₂And R₃When the catalyst is not hydrogen at the same time,R₁、R₂and R₃Not simultaneously being acetyl, R₁And R₂Is not benzyl at the same time; in formula II, R₁And R₂Not hydrogen or benzyl at the same time.
The drug having an anti-inflammatory bowel disease action according to claim 1, wherein said drug has any one of the following compound a-J structures:
a process for the preparation of a medicament having anti-inflammatory bowel disease activity having the structure shown in formula I, which comprises:

carrying out substitution reaction on myricetin shown in a formula III and a nucleophilic reagent in the presence of an alkaline reagent to obtain a compound shown in a formula I, wherein the reaction formula is as follows:

wherein R is₁And R₂Each independently selected from any one of hydrogen, ethyl, n-propyl, n-butyl, isopropyl, allyl, isobutyl, tert-butyl, benzyl, acetyl, sulfonic group, phosphoric group, benzoic group, benzamide group, benzoyl cyclopropylamine group, benzenesulfonic group, pyridine carboxamide cyclopropylamine group, pyrimidine carboxamide group or pyrimidine carboxamide cyclopropylamine group;

R₃any one selected from hydrogen, acetyl, sulfonic acid group or phosphoric acid group; and is

Satisfy R₁、R₂And R₃Not simultaneously being hydrogen, R₁、R₂And R₃Not simultaneously being acetyl, R₁And R₂Not benzyl at the same time.
The preparation method according to claim 3, wherein the compound represented by the formula III comprises myricetin and a nucleophilic reagent in a molar ratio of 1 to (1-6);

preferably, the solvent for the substitution reaction comprises any one of dichloromethane, tetrahydrofuran, acetonitrile, pyridine or triethylamine or a combination of at least two of the above;

preferably, the temperature of the substitution reaction is 0 to 80 ℃;

preferably, the time of the substitution reaction is 1 to 30 hours;

preferably, the basic reagent comprises any one or a combination of at least two of pyridine, triethylamine, N-diisopropylethylamine, N-methylmorpholine, potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide or potassium tert-butoxide, preferably pyridine or triethylamine.
A process for the preparation of a medicament having anti-inflammatory bowel disease activity having the structure shown in formula II, which comprises:

carrying out oxidation reaction on the compound shown in the formula IV in the presence of an oxidant to obtain a compound shown in a formula II, wherein the reaction formula is as follows:

wherein R is₁And R₂Each independently selected from any one of hydrogen, ethyl, n-propyl, n-butyl, isopropyl, allyl, isobutyl, tert-butyl, benzyl, acetyl, sulfonic group, phosphoric group, benzoic group, benzamide group, benzoyl cyclopropylamine group, benzenesulfonic group, pyridine carboxamide cyclopropylamine group, pyrimidine carboxamide group or pyrimidine carboxamide cyclopropylamine group; and is

Satisfy R₁And R₂Not hydrogen or benzyl at the same time.
The preparation method of claim 5, wherein the molar ratio of the compound shown in the formula IV to the oxidant is 1: (0.5-5);

preferably, the oxidant comprises any one or a combination of at least two of pyridinium chlorochromate, chromium trioxide, ozone, hydrogen peroxide or sulfur trioxide;

preferably, the solvent for the oxidation reaction comprises any one of dichloromethane, tetrahydrofuran, toluene, N-dimethylformamide, N-heptane or dimethyl sulfoxide or a combination of at least two thereof;

preferably, the temperature of the oxidation reaction is 0-50 ℃;

preferably, the time of the oxidation reaction is 1 to 10 hours.
The pharmaceutically acceptable salt of the drug having an anti-inflammatory bowel disease action according to claim 1 or 2.
The pharmaceutically acceptable salt of claim 7, wherein the pharmaceutically acceptable salt is a metal salt of a compound of formula I or a metal salt of a compound of formula II;

preferably, the metal salt comprises any one of a lithium salt, a sodium salt, a potassium salt, a magnesium salt or a calcium salt, preferably a sodium salt or a potassium salt.
The solvate of the drug having an anti-inflammatory bowel disease action according to claim 1 or 2.
The solvate according to claim 9, wherein the solvate is a hydrate of the compound of formula I, an alcoholate of the compound of formula I, a hydrate of the compound of formula II or an alcoholate of the compound of formula II.
The tautomer or the stereochemical isomer of the drug having an anti-inflammatory bowel disease action according to claim 1 or 2.
The prodrug of the drug having an anti-inflammatory bowel disease action according to claim 1 or 2.
A pharmaceutical composition comprising the drug having an anti-inflammatory bowel disease effect according to claim 1 or 2.
The pharmaceutical composition of claim 13, wherein the pharmaceutical composition further comprises an adjuvant;

preferably, the adjuvant comprises any one or a combination of at least two of an excipient, a diluent, a carrier, a flavoring agent, a binder, or a filler;

preferably, the dosage form of the anti-inflammatory bowel disease pharmaceutical composition includes any one of an oral preparation, a topical preparation or a parenteral preparation.
Use of the drug having an anti-inflammatory bowel disease action according to claim 1 or 2, the pharmaceutically acceptable salt of the drug having an anti-inflammatory bowel disease action according to claim 7 or 8, the solvate of the drug having an anti-inflammatory bowel disease action according to claim 9 or 10, the tautomer or the stereochemical isomer of the drug having an anti-inflammatory bowel disease action according to claim 11, the prodrug having an anti-inflammatory bowel disease action according to claim 12, or the pharmaceutical composition according to claim 13 or 14 for the preparation of a medicament for the treatment of inflammatory bowel disease.